Genomic deletion of Bcl6 differentially affects conventional dendritic cell subsets and compromises Tfh/Tfr/Th17 cell responses.

Conventional dendritic cells (cDC) play key roles in immune induction, but what drives their heterogeneity and functional specialization is still ill-defined. Here we show that cDC-specific deletion of the transcriptional repressor Bcl6 in mice alters the phenotype and transcriptome of cDC1 and cDC2, while their lineage identity is preserved. Bcl6-deficient cDC1 are diminished in the periphery but maintain their ability to cross-present antigen to CD8+ T cells, confirming general maintenance of this subset. Surprisingly, the absence of Bcl6 in cDC causes a complete loss of Notch2-dependent cDC2 in the spleen and intestinal lamina propria. DC-targeted Bcl6-deficient mice induced fewer T follicular helper cells despite a profound impact on T follicular regulatory cells in response to immunization and mounted diminished Th17 immunity to Citrobacter rodentium in the colon. Our findings establish Bcl6 as an essential transcription factor for subsets of cDC and add to our understanding of the transcriptional landscape underlying cDC heterogeneity.

TGF-ß specifies TFH versus TH17 cell fates in murine CD4+ T cells through c-Maf.

T follicular helper (TFH) cells are essential for effective antibody responses, but deciphering the intrinsic wiring of mouse TFH cells has long been hampered by the lack of a reliable protocol for their generation in vitro. We report that transforming growth factor-ß (TGF-ß) induces robust expression of TFH hallmark molecules CXCR5 and Bcl6 in activated mouse CD4+ T cells in vitro. TGF-ß-induced mouse CXCR5+ TFH cells are phenotypically, transcriptionally, and functionally similar to in vivo-generated TFH cells and provide critical help to B cells. The study further reveals that TGF-ß-induced CXCR5 expression is independent of Bcl6 but requires the transcription factor c-Maf. Classical TGF-ß-containing T helper 17 (TH17)-inducing conditions also yield separate CXCR5+ and IL-17A-producing cells, highlighting shared and distinct cell fate trajectories of TFH and TH17 cells. We demonstrate that excess IL-2 in high-density T cell cultures interferes with the TGF-ß-induced TFH cell program, that TFH and TH17 cells share a common developmental stage, and that c-Maf acts as a switch factor for TFH versus TH17 cell fates in TGF-ß-rich environments in vitro and in vivo.

Targeting intracellular and extracellular receptors with nano-to-macroscale biomaterials to activate immune cells.

Activation of immune cells is an essential process in innate and adaptive immunity. A high number of immune cell activation pathways have been discovered, which are stimulated via various intra- and extracellular receptors. Small-molecule and macromolecular agonists have been identified to target immune receptors in preclinical research and clinical practice. However, current immunostimulants are often associated with undesired side effects and/or low potency in vivo. These two issues have been addressed with multiscale biomaterials. In this review, we summarize and discuss the most explored intra/extracellular immune receptors which have been targeted with immunoactivating biomaterials. To target intracellular immune receptors, nano/microscale materials have been employed to deliver agonists into the endo/lysosomes or the cytoplasm. To target surface immune receptors, nano-to-macroscale biomaterials have been engineered to engage with them to activate immune cells. In this context, biomaterials are not only the drug carriers, but also function as part of the immunostimulants. The biomaterials-based modalities have shown clearly enhanced immunoactivation potency and decreased side effects compared to native immunostimulants. It is envisaged that nano-to-macroscale biomaterials will greatly contribute to the development of more effective strategies for immunoactivation, which have the potential to reshape future vaccination and immunotherapy.

T follicular helper cells in cancer.

T follicular helper (Tfh) cells provide essential help to B cells for effective antibody-mediated immune responses. Although the crucial function of these CD4+ T cells in infection and vaccination is well established, their involvement in cancer is only beginning to emerge. Increased numbers of Tfh cells in Tfh cell-derived or B cell-associated malignancies are often associated with an unfavorable outcome, whereas in various solid organ tumor types of non-lymphocytic origin, their presence frequently coincides with a better prognosis. We discuss recent advances in understanding how Tfh cell crosstalk with B cells and CD8+ T cells in secondary and tertiary lymphoid structures (TLS) enhances antitumor immunity, but may also exacerbate immune-related adverse events (irAEs) such as autoimmunity during immune checkpoint blockade (ICB) and cancer immunotherapy.

3D Tissue Explant and Single-Cell Suspension Organoid Culture Systems for Ex Vivo Drug Testing on Human Tonsil-Derived T Follicular Helper Cells.

Research on the human immune system is often restricted to peripheral blood cells. However, these cells can be different from those found in secondary lymphoid organs. For instance, specialized T and B cells that are localized in germinal centers (GCs), which are complex anatomical structures being required for the generation of potent antibodies, are not found in peripheral blood. Most T helper cells located in GCs belong to the T follicular helper (Tfh) cell subset, which provides critical support to B cells. Bona fide human GC Tfh cells can be obtained from secondary lymphoid tissues such as tonsils, which are routinely removed by surgery. We here describe a method that is based on human lymphoid histoculture (HLH) and human lymphoid aggregate culture (HLAC) to culture human adenoid (pharyngeal tonsil) tissue ex vivo, followed by deep Tfh cell phenotyping by flow cytometry. This method allows studying Tfh cells in a versatile explant culture system that preserves many aspects of the original in vivo three-dimensional (3D) structure, in parallel to single-cell suspension organoid cultures in which the original tissue structure is disintegrated. We also describe how this versatile platform can be used for drug testing or manipulation of human Tfh cells in vitro for mechanistic studies.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

Defining the RBPome of primary T helper cells to elucidate higher-order Roquin-mediated mRNA regulation.

Post-transcriptional gene regulation in T cells is dynamic and complex as targeted transcripts respond to various factors. This is evident for the Icos mRNA encoding an essential costimulatory receptor that is regulated by several RNA-binding proteins (RBP), including Roquin-1 and Roquin-2. Here, we identify a core RBPome of 798 mouse and 801 human T cell proteins by utilizing global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS). The RBPome includes Stat1, Stat4 and Vav1 proteins suggesting unexpected functions for these transcription factors and signal transducers. Based on proximity to Roquin-1, we select ~50 RBPs for testing coregulation of Roquin-1/2 targets by induced expression in wild-type or Roquin-1/2-deficient T cells. Besides Roquin-independent contributions from Rbms1 and Cpeb4 we also show Roquin-1/2-dependent and target-specific coregulation of Icos by Celf1 and Igf2bp3. Connecting the cellular RBPome in a post-transcriptional context, we find contributions from multiple RBPs to the prototypic regulation of mRNA targets by individual trans-acting factors.

Loss of direct adrenergic innervation after peripheral nerve injury causes lymph node expansion through IFN-γ.

Peripheral nerve injury can cause debilitating disease and immune cell-mediated destruction of the affected nerve. While the focus has been on the nerve-regenerative response, the effect of loss of innervation on lymph node function is unclear. Here, we show that the popliteal lymph node (popLN) receives direct neural input from the sciatic nerve and that sciatic denervation causes lymph node expansion. Loss of sympathetic, adrenergic tone induces the expression of IFN-γ in LN CD8 T cells, which is responsible for LN expansion. Surgery-induced IFN-γ expression and expansion can be rescued by ß2 adrenergic receptor agonists but not sensory nerve agonists. These data demonstrate the mechanisms governing the pro-inflammatory effect of loss of direct adrenergic input on lymph node function.

T follicular helper cells: linking cancer immunotherapy and immune-related adverse events.

Cancer immunotherapy utilizing immune checkpoint inhibitors (ICIs) has revolutionized the treatment of numerous cancer types. As the underlying mechanism of these treatments lies in the interference with inhibitory signals that usually impair potent antitumor immunity, for example, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and the programmed cell death protein 1 (PD-1):programmed death-ligand 1/2 (PD-L1/2) pathway, it is not surprising that this could also promote exaggerated adaptive immune responses to unrelated antigen specificities. One of the side effects of ICI-based cancer immunotherapy that is increasingly observed in the clinic is immune-related adverse events (irAEs), including various types of autoimmunity. However, the precise etiology is incompletely understood. T follicular helper (Tfh) cells provide essential help to B cells for potent antibody responses and their tumor tissue presence is often correlated with a better outcome in several solid tumor entities. Importantly, these CD4+ T cells express very high amounts of PD-1 and other co-stimulatory and inhibitory receptors. Here, we address the hypothesis that targeting CTLA-4 or PD-1 and its ligand PD-L1 critically impacts the function of Tfh cells in patients that receive these ICIs, thereby providing a link between ICI treatment and the development of secondary autoimmunity.

Antigen-dependent multistep differentiation of T follicular helper cells and its role in SARS-CoV-2 infection and vaccination.

T follicular helper (Tfh) cells play an essential role in regulating the GC reaction and, consequently, the generation of high-affinity antibodies and memory B cells. Therefore, Tfh cells are critical for potent humoral immune responses against various pathogens and their dysregulation has been linked to autoimmunity and cancer. Tfh cell differentiation is a multistep process, in which cognate interactions with different APC types, costimulatory and coinhibitory pathways, as well as cytokines are involved. However, it is still not fully understood how a subset of activated CD4+ T cells begins to express the Tfh cell-defining chemokine receptor CXCR5 during the early stage of the immune response, how some CXCR5+ pre-Tfh cells enter the B-cell follicles and mature further into GC Tfh cells, and how Tfh cells are maintained in the memory compartment. In this review, we discuss recent advances on how antigen and cognate interactions are important for Tfh cell differentiation and long-term persistence of Tfh cell memory, and how this is relevant to the current understanding of COVID-19 pathogenesis and the development of potent SARS-CoV-2 vaccines.

Continued Bcl6 Expression Prevents the Transdifferentiation of Established Tfh Cells into Th1 Cells during Acute Viral Infection.

T follicular helper (Tfh) cells are crucial for the establishment of germinal centers (GCs) and potent antibody responses. Nevertheless, the T cell-intrinsic factors that are required for the maintenance of already-established Tfh cells and GCs remain largely unknown. Here, we use temporally guided gene ablation in CD4+ T cells to dissect the contributions of the Tfh-associated chemokine receptor CXCR5 and the transcription factor Bcl6. Induced ablation of Cxcr5 has minor effects on the function of established Tfh cells, and Cxcr5-ablated cells still exhibit most of the features of CXCR5+ Tfh cells. In contrast, continued Bcl6 expression is critical to maintain the GC Tfh cell phenotype and also the GC reaction. Importantly, Bcl6 ablation during acute viral infection results in the transdifferentiation of established Tfh into Th1 cells, thus highlighting the plasticity of Tfh cells. These findings have implications for strategies that boost or restrain Tfh cells and GCs in health and disease.

Complex human adenoid tissue-based ex vivo culture systems reveal anti-inflammatory drug effects on germinal center T and B cells.

BACKGROUND: Human immunology research is often limited to peripheral blood. However, there are important differences between blood immune cells and their counterparts residing in secondary lymphoid organs, such as in the case of germinal center (GC) T follicular helper (Tfh) cells and GC B cells. METHODS: We developed a versatile ex vivo lymphoid organ culture platform that is based on human pharyngeal tonsils (adenoids) and allows for drug testing. We systematically phenotyped Tfh and GC B cell subsets in explant- and suspension cultures using multicolor flow cytometry and cytokine multiplex analysis. FINDINGS: Phenotypic changes of certain ex vivo cultured immune cell subsets could be modulated by cytokine addition. Furthermore, we optimized an activation-induced marker assay to evaluate the response to T cell stimulation. We provide proof-of-concept that Tfh and GC B cells could be modulated in these cultures by different anti-inflammatory drugs in unstimulated states and upon activation with vaccine-derived antigens. For example, GC B cells were lost upon CD40L blockade, and clinically approved JAK inhibitors impacted Tfh and GC B cells, including down-regulation of their key transcription factor BCL6. BCL6 regulation was affected by IL-6 signaling in T cells and IL-4 in B cells, respectively. Furthermore, we demonstrated that JAK signaling and TNF signaling contributed to the stimulation-induced activation of tonsil-derived T cells. INTERPRETATION: Our optimized methods, assays, and mechanistic findings can contribute to a better understanding of human GC responses. These insights may be relevant for improving autoimmune disease therapy and vaccination efficacy. FUNDING: This work was supported by a project grant under the joint research cooperation agreement of LMU Munich, LMU University Hospital, and Sanofi-Aventis Deutschland GmbH, as well as by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Emmy Noether Programme BA 5132/1-1 and BA 5132/1-2 (252623821), SFB 1054 Project B12 (210592381), and SFB 914 Project B03 (165054336).

Fingolimod Profoundly Reduces Frequencies and Alters Subset Composition of Circulating T Follicular Helper Cells in Multiple Sclerosis Patients.

Fingolimod is an effective treatment for relapsing-remitting multiple sclerosis. It is well established that fingolimod, a modulator of the sphingosine-1-phosphate pathway, restrains the egress of CCR7+ lymphocytes from lymphatic tissues into the blood, thus resulting in reduced lymphocyte counts in peripheral blood. CXCR5+ T follicular helper (Tfh) cells provide help to B cells, are essential for the generation of potent Ab responses, and have been shown to be critically involved in the pathogenesis of several autoimmune diseases. Besides lymphoid tissue-resident Tfh cells, CXCR5+ circulating Tfh (cTfh) cells have been described in the blood, their numbers correlating with the magnitude of Tfh cells in lymphoid tissues. Although the effect of fingolimod on circulating lymphocyte subsets has been established, its effect on cTfh cells remains poorly understood. In this study, we found that although fingolimod strongly and disproportionally reduced cTfh cell frequencies, frequencies of activated cTfh cells were increased, and the composition of the cTfh cell pool was skewed toward a cTfh1 cell phenotype. The circulating T follicular regulatory cell subset and CXCR5+ CD8+ T cell frequencies were also strongly and disproportionally decreased after fingolimod treatment. In contrast, relative frequencies of CXCR5- memory Th cells as well as regulatory T and B cells were increased. In summary, these data provide new insights into fingolimod-induced compositional changes of lymphocyte populations in the blood, in particular cTfh cells, and thus contribute to a better understanding of the mechanism of action of fingolimod in multiple sclerosis patients.

Diverse functions of miR-17-92 cluster microRNAs in T helper cells.

T helper (Th) cells are critically involved in adaptive immune responses against various pathogens. In contrast, dysregulated T helper cell responses are associated with a variety of diseases, including autoimmunity, allergies, and cancer. Differentiation of naïve CD4+ T cells into effector T helper cell subsets, including Th1, Th2, Th17, Treg, and T follicular helper (Tfh), requires precise dosing of signaling molecules and transcription factors. MicroRNAs (miRNAs), which are small endogenously expressed RNAs that regulate gene expression, play important roles in these processes. The miR-17-92 cluster, a miRNA polycistron also known as oncomiR-1, has emerged as a central integrator of gene expression events that govern T helper cell differentiation pathways. The complexity of miR-17-92-mediated gene regulation lies in the nature of this miRNA cluster, which consists of six different miRNAs. Individual miR-17-92 miRNAs, albeit initially transcribed as one transcript, can have cooperative or opposing effects on biological processes. Therefore, a better understanding of the molecular regulation of miR-17-92 and its downstream networks will provide important insights into T helper cell differentiation and diversity that may be harnessed for the design of advanced T cell-targeting therapies.

Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells.

Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.

MicroRNA regulation of type 2 innate lymphoid cell homeostasis and function in allergic inflammation.

MicroRNAs (miRNAs) exert powerful effects on immunity through coordinate regulation of multiple target genes in a wide variety of cells. Type 2 innate lymphoid cells (ILC2s) are tissue sentinel mediators of allergic inflammation. We established the physiological requirements for miRNAs in ILC2 homeostasis and immune function and compared the global miRNA repertoire of resting and activated ILC2s and T helper type 2 (TH2) cells. After exposure to the natural allergen papain, mice selectively lacking the miR-17∼92 cluster in ILC2s displayed reduced lung inflammation. Moreover, miR-17∼92-deficient ILC2s exhibited defective growth and cytokine expression in response to IL-33 and thymic stromal lymphopoietin in vitro. The miR-17∼92 cluster member miR-19a promoted IL-13 and IL-5 production and inhibited expression of several targets, including SOCS1 and A20, signaling inhibitors that limit IL-13 and IL-5 production. These findings establish miRNAs as important regulators of ILC2 biology, reveal overlapping but nonidentical miRNA-regulated gene expression networks in ILC2s and TH2 cells, and reinforce the therapeutic potential of targeting miR-19 to alleviate pathogenic allergic responses.

Experimental priming of encephalitogenic Th1/Th17 cells requires pertussis toxin-driven IL-1ß production by myeloid cells.

CD4(+) Th17 are heterogeneous in terms of cytokine production and capacity to initiate autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE). Here we demonstrate that experimental priming of encephalitogenic Th cells expressing RORγt and T-bet and producing IL-17A, IFN-γ and GM-CSF but not IL-10 (Th1/Th17), is dependent on the presence of pertussis toxin (PTX) at the time of immunization. PTX induces early production of IL-1ß by CD11b(+)CCR2(+)Gr1(+) myeloid cells, which are rapidly recruited to antigen-draining lymph nodes. PTX-induced generation of Th1/Th17 cells is impaired in IL-1ß- and ASC-deficient mice and in mice in which myeloid cells are depleted or fail to migrate to lymph nodes and requires expression of IL-1R1 and MyD88 on both T cells and non-T cells. Collectively, these data shed light on the enigmatic function of PTX in EAE induction and suggest that inflammatory monocytes and microbial infection can influence differentiation of pathogenic Th1/Th17 cells in autoimmune diseases through production of IL-1ß.

Self-enforcing feedback activation between BCL6 and pre-B cell receptor signaling defines a distinct subtype of acute lymphoblastic leukemia.

Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.

MicroRNA regulation of the germinal center response.

The generation of germinal centers (GCs) is a hallmark feature of the adaptive immune response, resulting in the production of high-affinity antibodies that neutralize pathogens and confer protection upon reinfection. The GC response requires interactions between different immune cell types, and the coordination of complex and dynamic gene expression networks within these cells. Here we provide deeper insights into how microRNAs, small endogenously expressed RNAs, regulate the cellular processes involved in the differentiation and function of T follicular helper cells and germinal center B cells, the two main players of the T cell-dependent humoral immune response.

Persistent antigen and germinal center B cells sustain T follicular helper cell responses and phenotype.

T follicular helper (Tfh) cells provide help to B cells and are crucial for establishment of germinal center (GC) reactions, including production of high-affinity antibodies and generation of memory B cells and long-lived plasma cells. Here we report that the magnitude of the Tfh cell response was dictated by the amount of antigen and directly correlated with the magnitude of the GC B cell response. In addition, maintenance of the Tfh cell phenotype required sustained antigenic stimulation by GC B cells. In lymphopenic conditions, a strong and prolonged Tfh cell response led to bystander B cell activation, hypergammaglobulinemia, and production of poly- and self-reactive antibodies. These data demonstrate that antigen dose determines the size and duration of the Tfh cell response and GC reaction, highlight the transient nature of the Tfh cell phenotype, and suggest a link between overstimulation of Tfh cells and the development of dysregulated humoral immune responses.